**3.7 The effect of fermentation components on isomerization reaction**

Red grape juice contains almost equal amounts of glucose and fructose before fermentation. Therefore, to determine enzyme activity in grape juices, 5% w/v fructose was added into grape juice. As a result, glucose and fructose concentrations remained stable for 2.5 days. The fructose concentrations of samples were higher than those of glucose. Although samples were kept for 2.5 days, the enzyme did not isomerize the substrate in solutions (data not shown). As a result, since enzyme activity was not observed in grape must before fermentation, it was thought that a component that is present before fermentation would be inhibiting the enzyme.

#### **3.8 The effect of calcium content on isomerization reaction**

Generally, red wine contains both calcium and magnesium in amounts of 80 ppm and 120 ppm, respectively [15]. The ratio of magnesium to calcium must be equal to 12 to provide activating conditions for glucose isomerase. It was thought that the inhibition in the wine medium was because of the calcium content. In light of this information, experiments were conducted in synthetic media containing ethanol, glycerol and calcium, and red wine with the increased magnesium content. The conversion reaction took place in a synthetic medium without calcium; however, there was no formation of glucose in the synthetic medium with calcium (data not shown). As a result of this experiment, it was thought that calcium in wine may be inhibiting the glucose isomerase.

After the addition of 840 ppm magnesium into red wine, there was no formation of glucose in the red wine medium even with increased magnesium content at pH 3.28. The glucose formation was observed in red wine media with increased pH with or without additional magnesium. However, the reaction rate was faster at pH 7.5 than at pH 8.0 since the medium at pH 7.5 contained additional magnesium. The reactions reached equilibrium after about 50 hours (data not shown). As a result, it was concluded that the additional magnesium had no effect on low pH wine medium; however, it speeded the reaction rate at high pH wine media. Therefore, the effect of pH on isomerization reaction in wine media must be considered.

Another experiment was conducted by considering the ion retention capacity of EDTA to hold the calcium in homemade red wine media. There was no formation of glucose after 70 hours (data not shown). Therefore, it was concluded that EDTA had no positive effect on isomerization reaction in red wine media.

To test the enzyme activity in red wine media containing little amounts of calcium, samples 0, 4, 6, and 10 were chosen. The glucose concentrations of samples 0, 4, 6, and 10 remained stable and isomerization reaction did not take place after 80 hours (data not shown). Whereas calcium in synthetic medium inhibited the activity of glucose isomerase, there was also an inhibition effect in red wine even if there was no calcium in the environment.

#### **3.9 The activity of glucose isomerase in dilute wine media**

In the first experiment, homemade red wines were diluted with distilled water at six different concentrations. As seen in **Table 1**, except for concentrations at 20 and 5% v/v, the glucose concentrations of samples remained stable. At concentrations of 5 and 20% v/v red wines, the glucose formation took place during incubation.

Also, different brands and types of wines at different dilution concentrations were used in another experiment. As seen in **Table 1**, the glucose formation took place at concentrations of 10 and 5% v/v regardless of the brand type. Also, almost *Determining Glucose Isomerase Activity in Different Wine Environments to Prevent Sluggish… DOI: http://dx.doi.org/10.5772/intechopen.100499*


**Table 1.** *Glucose concentrations of different wines with different dilution rates.* equal amounts of glucose formed at the same hours for different dilution factors and brand types.

As a result of these experiments, it was thought that a component or some components in wine coming from grapes may inhibit the isomerization reaction but this component or components may lose its or their effects in diluted wines but only at the levels of fivefold dilutions.

#### **3.10 The effect of tartaric acid on the enzyme activity**

The experiments were conducted with synthetic media containing fructose and tartaric acid and red wine containing no tartaric acid.

Fructose concentrations remained stable at 1% w/v at pH 3.55 and also, no glucose formation took place in flasks after 150 hours. However, glucose formation and fructose consumption were observed at pH 6.33. The reaction reached equilibrium within 30 hours with higher fructose contents (data not shown). As a result, L- (+)-tartaric acid inhibited the activity of glucose isomerase at pH 3.55; however, if the pH of the solution was 6.33, there was no inhibition with tartaric acid. Therefore, it was thought that if tartaric acid in wine is eliminated, the reaction would take place.

For experiments, samples numbered as 0, 2, 5, 8, 9, 10, and 12 were chosen. As seen in **Figure 3**, the 100% glucose formation took place at pH values of 9.85 and 6.7 and 50% glucose formation took place at 4.8, that is, sample numbers of 2, 5, and 8, respectively. Even if all samples did not contain tartaric acid, the conversion reaction took place at only higher pH values. Therefore, it was thought that the tartaric acid had an inhibitory effect at low pH values, under a pH of approximately 5. As a result, it had to be examined that glucose isomerase could or not convert fructose to glucose in wine media containing tartaric acid at high pH.

#### **3.11 The effect of pH on isomerization reactions in red wine medium**

As seen in **Figure 4**, the glucose formation took place at pH values of 6, 7, and 8 regardless of temperature. If temperature values were compared, the glucose concentration increased from 0.2% to almost 0.9% w/v for 60°C; however, it was from 0.2% to almost 0.4% w/v for 30°C during 70 hours of incubation. If pH values were compared, the reaction rates were higher for pH 8, 7, and 6 in decreasing order at 60°C. However, the reaction rate was almost equal for pH 7 and 8 but lower for pH 6 at 30°C.

**Figure 3.** *Glucose concentrations of wine samples without tartaric acid.*

*Determining Glucose Isomerase Activity in Different Wine Environments to Prevent Sluggish… DOI: http://dx.doi.org/10.5772/intechopen.100499*

**Figure 4.** *Glucose concentrations of homemade red wines at different pH values at 60 and 30°C.*

From the results of these experiments, the isomerization reactions took place in red wine media at pH values higher than 5 regardless of temperature. Therefore, it was thought that the pH of the medium had to be suitable in order for the glucose isomerase to be active in wine media.
